A number of clinical assays have been designed to measure the degree of turnover of the blood clotting system. These assays are routinely used to assess the severity of coagulopathies such as disseminated intravascular coagulation (DIC). Many of these assays are also being investigated for their utility in predicting hypercoagulable states that may predispose a person to developing deep vein thrombosis (DVT), heart attack, ischemic stroke, or other thrombotic and thromboembolic disorders. Examples of such assays are measurements of the plasma levels of fibrinopeptide A, thrombin-antithrombin complexes, prothrombin fragment 1+2, D-dimer, and factor VIIa (Amiral, J. and Fareed, J. (1966). “Thromboembolic diseases: biochemical mechanisms and new possibilities of biological diagnosis,” Semin Thromb Hemost 22 Suppl, 1:41-48; Gouin-Thibault, I. and Samama, M. M. 1999. “Laboratory diagnosis of the thrombophilic state in cancer patients,” Semin Thromb Hemost 25: 167-172; Morrissey, J. H. et al. 1993. “Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation,” Blood 81:734-744; and Morrissey, J. H. 1996. “Plasma factor VIIa: Measurement and potential clinical significance,” Haemostasis 26:66-71). These assays permit insights into the degree of ongoing activation of the blood clotting system in vivo. For example, the levels of fibrinopeptide A and D-dimer reflect the degree to which fibrinogen is being converted into fibrin. The levels of thrombin-antithrombin complexes and prothrombin fragment 1+2 reflect the degree to which prothrombin is being converted to thrombin in vivo. One cannot measure thrombin activity levels in plasma directly because the active enzyme has an extremely short plasma half-life (owing to plasma's high content of protease inhibitors). Thus, the levels of thrombin-antithrombin complexes reflect the ongoing rate of thrombin activation in vivo because antithrombin (formerly known as antithrombin III or ATIII) is a major inhibitor of thrombin in plasma, and because these complexes have a much longer half-life in plasma than does thrombin. Similarly, prothrombin fragment 1+2 is released from prothrombin when it is activated to thrombin, and these fragments can circulate at measurable levels in plasma. For these reasons, measurements of either thrombin-antithrombin complexes or prothrombin fragment 1+2 in plasma are thought to reflect the ongoing rate of thrombin generation in vivo.
Assays to measure plasma markers of activation of other blood clotting factors have also been developed, in addition to those specific for the activation of thrombin. In the case of factors IX and X, as with thrombin, the active proteases (factors IXa and Xa) have very short half-lives in plasma, so it is not possible to measure their levels directly. However, as with prothrombin, assays have been developed to indirectly assess the degree of activation of factors IX and X. One set of methods involves measuring the plasma levels of activation peptides released from factors IX or X when they are converted to factors IXa or Xa. These activation peptides circulate with much longer half-lives than do the activated proteases themselves, and assays based on measuring the levels of such activation peptides have been developed and applied in a variety of epidemiologic studies (Bauer, K. A. 1994. “New markers for in vivo coagulation,” Curr Opin Hematol 1:341-346; Bauer, K. A. 1999. “Activation markers of coagulation,” Baillieres Best Pract Res Clin Haematol 12:387-406; and Cooper, J. A. et al. 2000. “Comparison of novel hemostatic factors and conventional risk factors for prediction of coronary heart disease,” Circulation 102:2816-2822).
Another way to assess the ongoing rate of activation of factors IX or X is to measure the levels of circulating complexes of factor IXa or factor Xa with their plasma inhibitors. This has been done for factor IXa-antithrombin complexes (Takahashi, et al. 1991. “Activated factor IX-antithrombin III complexes in human blood: quantification by an enzyme-linked differential antibody immunoassay and determination of the in vivo half-life,” J Lab Clin Med 118:317-325) and factor Xa-antithrombin complexes (Gouin-Thibault, I. et al. 1995. “Measurement of factor Xa-antithrombin III in plasma: relationship to prothrombin activation in vivo,” Br J Haematol 90:669-680; Bauer, K. A. 1994. “New markers for in vivo coagulation,” Curr Opin Hematol 1:341-346; and Bauer, K. A. 1999. “Activation markers of coagulation,” Baillieres Best Pract Res Clin Haematol 12:387406). In addition, assays for measuring the circulating levels of complexes between factor Xa and tissue factor pathway inhibitor (TFPI) have also been developed (Okugawa, Y. et al. 2000. “Increased plasma levels of tissue factor pathway inhibitor-activated factor X complex in patients with disseminated intravascular coagulation,” Am J Hematol 65:210-214; Iversen, N. et al. 2000. “Tissue factor pathway inhibitor (TFPI) in disseminated intravascular coagulation: low levels of the activated factor X-TFPI complex,” Blood Coagul Fibrinolysis 11:591-598; Iversen, N. et al. 2000. “Tissue factor pathway inhibitor (TFPI) in disseminated intravascular coagulation: low levels of the activated factor X-TFPI complex,” Blood Coagul Fibrinolysis 11:591-598; and Miller, G. J. 2000. “Haemostatic factors in human peripheral afferent lymph,” Thromb Haemost 83:427-432).
Coagulation factor VII is converted to the activated form, factor VIIa, by proteolysis of a single peptide bond, and this is not associated with the release of an activation peptide (Morrissey, J. H. 2001. “Tissue factor and factor VII initiation of coagulation,” in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, R. W. Colman et al. eds., Philadelphia: Lippincott Williams & Wilkins, pp. 89-101). For this reason, it is not possible to monitor factor VII activation using an activation peptide assay, as has been done with prothrombin or factors IX or X. However, factor VIIa has a relatively long plasma half-life (about 2 hours), and so it has been possible to develop a clotting assay (based on a mutant form of tissue factor) that can specifically measure the levels of active factor VIIa in plasma (Morrissey, J. H. et al. 1993. “Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation,” Blood 81:734-744).
Measuring the plasma levels of factor VIIa are of interest because the blood clotting system is initiated when factor VII or VIIa binds to tissue factor (an integral membrane protein) on cell surfaces. The resulting membrane-bound complex of factor VIIa and tissue factor is the most potent known initiator of blood clotting. Tissue factor is normally present only on cells outside the vasculature, and it triggers blood clotting in normal hemostasis following vascular injury, thereby allowing blood to come into contact with tissue factor. Tissue factor expression can be induced on monocytes and endothelial cells by inflammatory mediators. Induced expression of tissue factor is thought to be responsible for the pathologic activation of blood clotting that triggers a number of thrombotic disorders (Morrissey, J. H. 2001. “Tissue factor and factor VII initiation of coagulation,” in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, R. W. Colman et al. eds., Philadelphia: Lippincott Williams & Wilkins, pp. 89-101).
Factor VIIa has a long half-life in plasma because it is essentially unreactive with any of the plasma protease inhibitors in the absence of its cofactor, tissue factor (Morrissey, J. H. 2001. “Tissue factor and factor VII initiation of coagulation,” in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, R W. Colman et al. eds., Philadelphia: Lippincott Williams & Wilkins, pp. 89-101). However, when factor VIIa binds to tissue factor, it becomes susceptible to inhibition both by antithrombin and TFPI. Interestingly, when factor VIIa bound to tissue factor reacts with antithrombin, the resulting factor VIIa-antithrombin (factor VIIa-AT) complexes lose affinity for tissue factor. Consequently these complexes, once formed, are released into solution (Hamamoto, T. and Kisiel, W. 1998. “The effect of cell surface glycosaminoglycans (GAGS) on the inactivation of factor VIIa-tissue factor activity by antithrombin III,” Int J Hematol 68:67-78; Kondo, S. and Kisiel, W. 1987. “Regulation of factor VIIa in plasma: Evidence that antithrombin III is the sole plasma proteinase inhibitor of human factor VIIa,” Thromb Res 46:325; Lawson, J. H. et al. 1993. “Complex-dependent inhibition of factor VIIa by antithrombin III and heparin,” J Biol Chem 268:767-770; and Rao, L. V. M. et al. 1993. “Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa,” Blood 81:2600-2607).
Because factor VIIa is only susceptible to inhibition by antithrombin when it is bound to tissue factor, and because the resulting factor VIIa-AT complexes are released from tissue factor, the circulating levels of factor VIIa-AT reflect the degree of exposure of tissue factor to the blood. Such intravascular exposure of tissue factor is expected to result from inflammatory states and in fact has previously been shown to drive the lethal coagulopathy associated with sepsis (De Boer, J. P. et al. 1993. “Activation patterns of coagulation and fibrinolysis in baboons following infusion with lethal or sublethal dose of Escherichia coli,” Circ Shock 39:59-67; Drake, T. A. et al. 1993. “Expression of tissue factor, thrombomodulin, and E-selectin in baboons with lethal Escherichia coli sepsis,” Am J Pathol 142:1458-1470b; and Taylor, F. B. et al. 1991. “Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody,” Circ Shock 33:127-134). In addition, ongoing intravascular exposure of tissue factor, possibly due to chronic inflammatory conditions, may contribute to hypercoagulable states that could lead to the development of thrombotic diseases. For these reasons, it is of interest to be able to estimate the level of intravascular exposure of tissue factor.
It has now been found that measurable levels of factor VIIa-AT complexes are found in plasma and can be used to estimate the level of intravascular exposure of tissue factor. An ELISA assay has been developed for measuring the levels of factor VIIa-AT complexes in plasma Antibodies for use in the ELISA and methods of using the assay to assess patient risk or monitor anticoagulant therapy are also disclosed.